Method for purifying tetracycline



7 147,471 Patented Aug. 12, 1958 ice METHOD FUR PURIFYING 'IE'DRACYCLINEJohn Vand'eputte, Milltown, and Leon J. Heuser, Princeton, N. J.,assignors to Olin Mathieson Chemical Corporation, New York, N. Y., acorporation of Virginia No Drawing. Application March 4, 1955 Serial No.492,295

6 Claims. (Cl. 260559) This invention relates to methods of purifyingantibiotics, and more particularly to a method for isolatingtetracycline from a fermentation broth or other aqueous mediumcontaining the antibiotic.

Prior to this invention, it had been shown that a potent antibiotic,called tetracycline, is present in the culture liquids obtained whensuitable strains of certain Streptomyces species are grown in or onliquid nutrient media (of, for example, Minieri et al., AntibioticAnnual, 1953-1954, 8l-87; and U. S. patent application of Donovick, etal., Serial No. 443,952, filed July 16, 1954, now abandoned). It wasfurther known that when tetracycline is produced by a fermentationprocess, the tetracycline formed is mixed with various (solid andliquid) genetic contaminants, often including a second antibiotic,chlortctracycline, formed concurrently. Before this invention, however,no simple and efficient method was known for separating the tetracyclinefrom the solids (e. g. the mycelia, spores, and other sedimentablesolids), other antibiotics concurrently formed, and inactive materialsin the culture medium.

An object of this invention, therefore, is the provision of an improvedmethod of recovering tetracycline from a fermentation broth containingit and genetic contaminants.

Another object of this invention is the provision of a method ofseparating tetracycline from other antibiotics produced concurrently ina fermentation process utilizing a tetracycline-producing Streptomyces.

Still another object of this invention is the provision of a method ofseparating tetracycline from inactive impurities present in a culturefiltrate which has previously been separated from the culture medium ina fermentation process involving a tetracycline-producing Streptomyces.

These objects are achieved by the method of this invention, whichessentially comprises adsorbing tetracycline from a filtrate of aculture of a tetracyclineproducing Streptomyces by treating the filtratewith an inorganic metal compound, and then recovering the tetracyclinefrom the compound. The inorganic metal compounds useful in the practiceof this invention are the relatively water-insoluble salts or bases(compounds having a solubility in water of less than about 0.005 a gramcompound per ml. water) of a polyvalent light metal, such as a lightmetal selected from the groups IIA and 1118 of Mendeleeffs periodictable. Metals whose salts and/ or bases fulfill the above requirementsinclude, inter alia, calcium, strontium, barium, and aluminum. Examplesof inorganic metal compounds suitable for the purposes of this inventioninclude the salts and bases of the alkaline earth metals [group HA] (e.g. calcium sulfate, calcium carbonate, strontium sulfate, bariumsulfate, barium carbonate and calcium oxide) and the salts and oxides ofthe non-heavy metals of group IIIA (e. g. aluminum oxide). Of these,calcium sulfate is preferred. The salts and bases must not be highlywater soluble, since the operability of the process depends on theability of particles of the inorganic metal compounds to adsorb andthereby remove the tetracycline from an aqueous medium. Throughout thespecification and claims this ability of particles of the inorganicmetal compounds to remove the tetracycline is referred to as anadsorption, but We do not Wish to be limited to any particular theoryfor the phenomenon; accordingly, the term adsorption is to be understoodto include complex-formation, occlusion, or any other mechanism wherebythe tetracycline is tied up with the inorganic metal compound andthereby removed from solution.

Preparatory to the addition of the inorganic metal compound, the wholeculture (broth) of a tetracyclineproducing Streptomyes is acidified to apH of less than about 2.5 (preferably about 2) with an acid such ashydrochloric acid, and the culture liquid is removed from the residualculture solids, as by filtering or centrifuging. The acidified culturefiltrate, which contains the main portion of the tetracycline in thebroth, is then treated with the inorganic metal compound. Either after,with, or prior to, the addition of the inorganic metal compound, the pHof the culture filtrate is adjusted to the basic side, preferably to arange of about 9 to 10 (optimally about 9.5), by treatment with a base,such as an alkali metal base (e. g. sodium hydroxide); and the resultingmixture is then filtered to separate the undissolved inorganic metalcompound and adsorbed tetracycline from the soluble inactive impuritiesin the culture filtrate. This insoluble association of inorganic metalcompound and tetracycline, which usually contains some water, will bereferred to hereinafter as the wet cake. The proportion of inorganicmetal compound to filtrate is not critical; but to insure completeadsorption of tetracycline, at least 1% by weight of compound per volumeof filtrate should be employed. This step in the process, as well as allsubsequent steps, can be carried out at normal temperatures such asambient temperature. In order to prevent any possible decomposition ofthe tetracycline, however, the temperature should not be permitted torise above about 35 C.

The tetracycline is recovered from the wet cake by elution therefrom.This elution is accomplished by slurrying the wet cake in an organicsolvent to form a slurry of wet organic solvent, the inorganic metalcompound and tetracycline. By wet organic solvent is meant a mixture ofa solvent and water wherein the water is present in the range of 5% to50% v./v. based on the total mixture, the requisite moisture usuallybeing supplied by the water content of the wet cake. Suitable organicsolvents utilizable in this step of the process are those whereintetracycline is soluble, and include alcohols, such as alkanols (c. g.methanol, isopropanol, butanol, and pentasol), ketones, such as acetoneand the cellosolves (e. g. methyl and ethyl), and esters such as thealkyl esters of lower fatty acids (e. g. amyl acetate). For this elutionstep, the pH of the slurry is adjusted to an acid value by means of anacid, such as a mineral acid (e. g. hydrochloric acid, sulfuric acid,and nitric acid). By this procedure, the tetracycline is desorbed fromthe inorganic metal compound and passes into solution in the wet organicsolvent.

To precipitate the metal cation of the adsorbing salt or base, the acidused in adjusting the pH of the slurry to said acid value is so chosenthat the anion of the acid forms a salt with the metal cation which isinsoluble in the wet solvent at the pH of the solution. Thus, if awater-immiscible solvent, such as butanol or pentasohis used as theorganic solvent, and a calcium salt or base is used as the inorganicmetal compound, then by adjusting the slurry to apH in the range ofabout 1.5 to 2.5 (preferably about 2) with sulfuric acid, insolublecalcium sulfate is formed, which may be removed byfiltration and canthereafter be recovered and reused in the purification.

and separation of tetracycline from a subsequent batch of culturefiltrate. trated by Example 1. it should be noted, however, that thisspecific process is merely exemplary, and it will be obvious to oneskilled in the art which acid and organic solvent should be used,depending on the nature of the inorganic metal compound used to adsorbthe tetracycline.

if a water-miscible solvent, such as methanol, ethanol, isopropanol, oraceto e, is used as the organic solvent, and a calcium salt base is usedas the inorganic metal compound, then by adjusting the slurry to a pH inthe range of about 1.5 to 2.5 (preferably about 2) with sulfuric acid,most of the calcium sulfate precipitates and can be removed byfiltration. Some of the calcium ion, however, remains in solution; andthis residual amount can be removed by forming a different salt ofcalcium, which is more insoluble in the wet organic solvent, as bytreatment with oxalic acid or a salt thereof (and, if necessary, adju-ing the pito precipitate the remaining calcium ion as calcium oxalate.This alternative in the process is illustrated by Example 2. it shouldbe noted here also, that the specific process is merely exemplary; andit will be obvious to one sltilled in the art as to which alternativeinorganic compounds, acids, and organic solvents may be used.

The wet organic solvent solution (filtrate), freed of the cation of theinorganic compound, is then adjusted to a pH of about to 4.5 (preferablyabout 4.2) by the addi tion of a base such as sodium hydroxide (and, ifnecessary, water to precipitate the tetracycline as the free base. Theprecipitate is then recovered by filtration.

The tetracycline can be further purified and converted to anacid-solution salt by dissolving the base in a wet organic solvent, suchas butanol, and treating the solution with the desired acid to a pH ofabout 1.5 to 2.5 (preferably about 2). The water and a portion of theorganic solvent are removed by azeotropic distillation und upon coolingto say 5 C., the acidvcline is recovered as a precipitate. practice orthis invention, not only is tetracycline isolated from the inactiveimpurities normally found in a fermentation broth but also from anychlortetracycline which may have been produced during fermentation.separation is made possible because chlortetrucycline is more soluble inthe organic solvents used the procoses of this invention than istetracycline, so that a substantial proportion of the chlortetracyclineremains in solution as the tetracycline is precipitated therefrom.Another advantage of this process re sides in the fact that throughoutmost of the treatment, the pH of the tetracyclinecontaining mixture ison the basic side. Tetracycline is more stable in a basic medium than inan acid medium, wherein it tends to dehydrate to anhydrotetracycline.This added advantage permits a larger recovery of tetracycline withconsequent decrease in conversion of tetracycline toanhydrotetracycline. Furthermore, since cldortetracycline is relativelyunstable in a highly basic medium, a substantial portion thereof isdestroyed in the practice of the method this invention.

The following examples are illustrative of the invention:

Example 1 One liter of a whole fermentation broth assaying 1200 u./ml.for tetracycline is adjusted to pH 2 using hydro This alternative in theprocess is illusml. butanol, the pH is lowered to 2 with concentratedsulfuric acid, the mixture is stirred for one-half hour, and theinsolubles are thenfiltered off. The spent calcium sulfate cake retainsabout 2% of the activity. To the butanol filtrate is then added 20 ml.Water, and the pH is raised with stirringto 4.2 using a sodium hydroxidesolution. After stirring two to three hours, the precipitatedtetracycline base is filtered off and dried. The product weighs about0.98 g. and assays 780 u./mg. for tetracycline, thereby representing ayield of about 64%.

Example 2 One liter of whole fermentation broth assaying 1050 u./ml. fortetracycline is acidified to pH 2, filtered, l0 c to 1m sulfate isadded, the pH is raised to 9.5, the :--.xtiu'e is stirred for one-halfhour, and the insolubles filtered off. The spent filtrate assays 62u./ml. for tetracycline, indicating that 94% of the activity isadsorbed. The wet calcium sulfate, cake is then slurried in 30 ml. 70%aqueous acetone, the pH lowered to 2 with sulfuric acid, and the mixtureis stirred for one-half hour and filtered. The spent calcium sulfateinsoluble-s contain about 4% of the activity. To the filtrate is added0.2 g. ammonium oxalate, the pH is adjusted to 3 with sodium hydroxide,and the precipitate of calcium oxalate is filtered off. The pH of thefiltrate is then raised to 4.2 by addition of sodium hydroxide, stirredthree or four hours, and the p ecipitated tetracycline base filtered offand dried. The product weighs about 0.84 g. and assays 720 u./ mg. fortetracycline, thereby representing a yield of about 59%.

Example 3 The crude tetracycline base formed by the procedure of eitherExample 1 or 2 can be purified and converted to its hydrochloride bydissolving l g. of the base in 26 ml. Water-saturated butanol, andadjusting the pH to 2 by addition of concentrated hydrochloric acid.After stirring one-half hour, the insolubles are filtered off and thefiltrate vacuum-distilled to a volume of 5 ml. On standing in a coldroom, the hydrochloride of tetracycline precipitates and is filteredoff, washed with a butano 50% hexane solution, then hexane alone anddried. The yield amounts to about -70% of a product assaying about900-920 u./mg.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

We claim:

1. A method of isolating tetracycline from a fermentation brothcontaining the same and appreciable quantities of chlortetracycline,which comprises acidifying said broth; filtering the broth; treating thefiltrate with an inorganic metal compound selected from the groupconsisting of the relatively water-insoluble salts and bases of thelight metals of groups HA and HIE of the periodic table at a pH in therange of about 9 to about 10, said compound being present in aconcentration greater than about 0.005 gram of compound per milliliterof filtrate, whereby an insoluble association of tetracycline and inorganic compound precipitates; separating the precipitate; mixing theprecipitate with an organic solvent for tetracycline thereby forming amixture comprising water, organic solvent, inorganic metal compound andtetracycline; acidifying the mixture to a pH in the range of about 1.5to about 2.5 with an acid which forms an insoluble salt with the cationof the inorganic metal compound in sai solvent at said acid pH; removingthe insoluble salt; and recovering the tetracycline from the solvent byadjusting the pH thereof to the range of about 4 to about 4.5, wherebytetracycline precipitates.

2. The method of claim 1 wherein the inorganic metal compound is calciumsulfate.

3. The method of claim 1 wherein the solvent is butanol.

4. The method of claim 3 wherein the inorganic metal compound is calciumsulfate and the acid is sulfuric acid.

5. A method of isolating tetracycline from a fermentation brothcontaining the same and appreciable quantities of chlortetracycline,which comprises acidifying said broth; filtering the broth; treating thefiltrate with an inorganic relatively water-insoluble calcium salt at apH in the range of about 9 to about 10, said compound being present in aconcentration greater than about 0.005 gram of compound per milliliterof filtrate whereby an insoluble association of tetracycline and calciumcompound precipitates; separating the precipitate; mixing theprecipitate with a water-miscible organic solvent for tetracyclinethereby forming a mixture comprising water, organic solvent, calciumcompound and tetracycline; acidifying the mixture to a pH in the rangeof about 1.5 to about 2.5 with an acid which forms an insoluble saltwith calcium in said solvent at said acid pH; removing the insolublesalt; treating the remaining solution with a compound selected from thegroup consisting of oxalic acid and salts thereof to precipitate anyremaining calcium ion as calcium oxalate; removing the precipitate; andrecovering the tetracycline from the solvent by adjusting the pH thereofto the range of about 4 to about 4.5, whereby tetracycline precipitates.

6. The method of claim 5 wherein the solvent is acetone and the acid issulfuric acid.

References Cited in the file of this patent UNITED STATES PATENTS2,609,329 Niedercorn Sept. 2, 1952 2,640,842 Weidenheimer et al June 2,1953 2,655,535 Pidacks et a1. Oct. 13, 1953 2,658,077 Harms Nov. 3, 19532,699,054 Conover Jan. 11, 1955 2,739,924 Lein et. a1 Mar. 27, 19562,804,476 Bogert Aug. 27, 1957 FOREIGN PATENTS 497,340 Canada Nov. 3,1953 497,485 Canada Nov. 3, 1953 504,172 Canada July 6, 1953 OTHERREFERENCES Bird, et al.: Antibiotics and Chemotherapy," vol. 4, No. 7,July 1954 (received Feb. 8, 1954), page 750.

1. A METHOD OF ISOLATING TETRACYCLINE FROM A FERMENTATION BROTHCONTAINING THE SAME AND APPRECIABLE QUANTITIES OF CHLORTETRACYCLINE,WHICH COMPRISES ACIDIFYING SAID BROTH; FILTERING THE BROTH; TREATING THEFILTRATE WITH AN INORGANIC METAL COMPOUND SELECTED FROM THE GROUPCONSISTING OF THE RELATIVELY WATER-INSOLUBLE SALTS AND BASES OF THELIGHT METALS OF GROUPS IIA AND IIIB OF THE PERIODIC TABLE AT A PH IN THERANGE OF ABOUT 9 TO ABOUT 10 SAID COMPOUND BEING PRESENT IN ACONCENTRATION GREATER THAN ABOUT 0.005 GRAM OF COMPOUND PER MILLILITEROF FILTRATE, WHEREBY AN INSOLUBLE ASSOCIATION OF TETRACYCLINE ANDINORGANIC COMPOUND PRECIPITATES; SEPARATING THE PRECIPITATE; MIXING THEPRECIPITATE WITH AN ORGANIC SOLVENT FOR TETRACYCLINE THEREBY FORMING AMIXTURE COMPRISING WATER, ORGANIC SOLVENT, INORGANIC METAL COMPOUND ANDTETRACYCLINE; ACIDIFYING THE MIXTURE TO A PH IN THE RANGE OF ABOUT 1.5TO ABOUT 2.5 WITH AN ACID WHICH FORMS AN INSOLUBLE SALT WITH THE CATIONOF THE INORGANIC METAL COMPOUND IN SAID SOLVENT AT SAID ACID PH;REMOVING THE INSOLUBLE SALT; AND RECOVERING THE TETRACYCLINE FROM THESOLVENT BY ADJUSTING THE PH THEREOF TO THE RANGE OF ABOUT 4 TO ABOUT4.5, WHEREBY TETRACYCLINE PRECIPITATES.